Dr. B Notes (090513):
determine best PDB to use for Homology Model - see Target Discovery protocol
create a Homology model using ICM - see GoogleDocs/Protocols/VirtualScreening/HomologyModel/
Instructions ProMol Project Homology Models v6
*Target (protein/gene name): Protein Phosphatase 2A *NCBI Gene # or RefSeq#: Tb927.3.1240 *Protein ID (NP or XP #) or Wolbachia#: *Organism (including strain): T. Brucei Etiologic Risk Group (see link below): Appendix B-II-C *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
The protozoan organism Trypanosoma brucei is responsible for the disease African Trypanosomiasis, also known as African Sleeping Sickness. The organism is considered a Risk Group 2 organism and is considered a neglected tropical disease, according to TDR Targets Database. The protist is transmitted through the bite of a tsetse fly and it is actually possible for a person to be infected with this disease yet show no sign of any symptoms until many months or years later. Without any treatment, this disease occurs in two stages: the first stage symptoms include fever, joint pain and itching and in the second stage includes infection to the central nervous system, causing changes in behavior, loss of certain motor functions and coordination. The damage to the central nervous system can affect normal sleep patterns and thus gives its name. There is currently no known drug to treat African Trypanosomiasis. Link to TDR Targets page (if present): http://tdrtargets.org/targets/view?gene_id=19960 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: essential Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: none -- For Homology Model option: 2nppF ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB used results from SWISS-MODEL 2nppF sequence: MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNY LFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVD GQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRA HQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRTPDYFL ---- Query Coverage: 71% ---- Max % Identities: % ---- % Positives: 84% ---- Chain used for homology: Chain F
Current Inhibitors: microcystin Expression Information (has it been expressed in bacterial cells): Purification Method: Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids: 323 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
36217.3 Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 43360 Abs 0.1% (=1 g/l) 1.197, assuming all pairs of Cys residues form cystines
Ext. coefficient 42860 Abs 0.1% (=1 g/l) 1.183, assuming all Cys residues are reduced TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): *GC% Content for gene: 54.7% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCCGGCGACCACCTCTATCCCGCCTGTGACTGCGCCTGGTACCGGTGGTGAGGCGTCT
TCCTTCAATGTTGACGAGCTCATCCAGTACGTTCTGCAGGGTAAACCGCTGTCTGAACCG
CAGGTTGCCCGTCTGTGCCAGAAAGCGCGTGAAGTTCTGGAAAAAGAAGAAAACGTGCAC
ACCGTTCGTGCGCCAGTTACCGTTTGCGGTGACATCCATGGTCAGTTCCACGATCTGCTG
GAACTCCTGAAAATTGGTGGTCTCCCACCGGACACCAACTATCTCTTCATGGGTGACTAC
GTTGATCGCGGCTACTACTCCGTTGAAACCGTTACGCTCCTCCTGCTGTTCAAAATCCGC
TACCCGGAGCGTGTTCAGATCCTGCGTGGTAATCATGAATCTCGTCAGATCACCCAGGTT
TACGGCTTCTACGACGAATGCGTTCGTAAATACGGTTCTGCGAATGTTTGGAAACTGCTG
ACGGATCTGTTCGACTACCTGCCGCTGGCGGCTGTTGTTGAAAACGAAATCTTCTGCCTG
CACGGTGGCCTGTCTCCGACCCTGGACTCTCTGGCTCACATCCGTTCTCTGGAACGTGTC
CAAGAGGTCCCGCATGAAGGCCCGATGTGCGACCTCCTCTGGTCCGATCCGGAAGACAAA
GACGGTTGGGGTATCTCTCCGCGTGGTGCGGGTTTCACCTTCGGTGCGGACATTACCGAA
CAGTTCTGCCACAACAATGGTCTGAAAACCATCGCGCGTGCTCACCAGCTCGTAGCCGAG
GGTTACTCTTGGGCGCACTCTGATAAACTGGTTACTATCTTCTCTGCGCCGAACTACTGC
TATCGTTGTGGTAACCTGGCGGGTCTGCTCGAACTCGACGAACACATGAACAAATGCTTC
TTTCAGTTCGACCCGGCACCTCGTCGCGGTGAAGCACAAGTCTCCAAGAAGACCCCGGAC
TACTTCCTGTAA
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
determine best PDB to use for Homology Model - see Target Discovery protocol
create a Homology model using ICM - see GoogleDocs/Protocols/VirtualScreening/HomologyModel/
Instructions ProMol Project Homology Models v6
*Target (protein/gene name): Protein Phosphatase 2A
*NCBI Gene # or RefSeq#: Tb927.3.1240
*Protein ID (NP or XP #) or Wolbachia#:
*Organism (including strain): T. Brucei
Etiologic Risk Group (see link below): Appendix B-II-C
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
The protozoan organism Trypanosoma brucei is responsible for the disease African Trypanosomiasis, also known as African Sleeping Sickness. The organism is considered a Risk Group 2 organism and is considered a neglected tropical disease, according to TDR Targets Database. The protist is transmitted through the bite of a tsetse fly and it is actually possible for a person to be infected with this disease yet show no sign of any symptoms until many months or years later. Without any treatment, this disease occurs in two stages: the first stage symptoms include fever, joint pain and itching and in the second stage includes infection to the central nervous system, causing changes in behavior, loss of certain motor functions and coordination. The damage to the central nervous system can affect normal sleep patterns and thus gives its name. There is currently no known drug to treat African Trypanosomiasis.
Link to TDR Targets page (if present):
http://tdrtargets.org/targets/view?gene_id=19960
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: essential
Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford
Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in bloodstream forms (6 days); Source study: alsford
Gene/Ortholog: Tb927.3.1240 (OG4_10586); Phenotype: significant loss of fitness in procyclic forms; Source study: alsford
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): 0.8
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
p-nitrophenyl phosphate + H2O
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/assay-library/ec-number-iii.html
-- -or link (or citation) to paper that contains assay information
http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=655670
-- links to assay reagents (substrates) pages.
http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=655670
--- List cost and quantity of substrate reagents, supplier, and catalog #
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: none
-- For Homology Model option: 2nppF
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
used results from SWISS-MODEL
2nppF sequence:
MDEKVFTKELDQWIEQLNECKQLSESQVKSLCEKAKEILTKESNVQEVRCPVTVCGDVHGQFHDLMELFRIGGKSPDTNY
LFMGDYVDRGYYSVETVTLLVALKVRYRERITILRGNHESRQITQVYGFYDECLRKYGNANVWKYFTDLFDYLPLTALVD
GQIFCLHGGLSPSIDTLDHIRALDRLQEVPHEGPMCDLLWSDPDDRGGWGISPRGAGYTFGQDISETFNHANGLTLVSRA
HQLVMEGYNWCHDRNVVTIFSAPNYCYRCGNQAAIMELDDTLKYSFLQFDPAPRRGEPHVTRRTPDYFL
---- Query Coverage: 71%
---- Max % Identities: %
---- % Positives: 84%
---- Chain used for homology: Chain F
Current Inhibitors: microcystin
Expression Information (has it been expressed in bacterial cells):
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MPATTSIPPVTAPGTGGEASSFNVDELIQYVLQGKPLSEPQVARLCQKAREVLEKEENVHTVRAPVTVCGDIHGQFHDLLELLKIGGLPPDT
NYLFMGDYVDRGYYSVETVTLLLLFKIRYPERVQILRGNHESRQITQVYGFYDECVRKYGSANVWKLLTDLFDYLPLAAVVENEIFCLHGGLS
PTLDSLAHIRSLERVQEVPHEGPMCDLLWSDPEDKDGWGISPRGAGFTFGADITEQFCHNNGLKTIARAHQLVAEGYSWAHSDKLVTIFSA
PNYCYRCGNLAGLLELDEHMNKCFFQFDPAPRRGEAQVSKKTPDYFL
*length of your protein in Amino Acids: 323
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
36217.3
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 43360 Abs 0.1% (=1 g/l) 1.197, assuming all pairs of Cys residues form cystines
Ext. coefficient 42860 Abs 0.1% (=1 g/l) 1.183, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene: 54.7%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGCCGGCGACCACCTCTATCCCGCCTGTGACTGCGCCTGGTACCGGTGGTGAGGCGTCT
TCCTTCAATGTTGACGAGCTCATCCAGTACGTTCTGCAGGGTAAACCGCTGTCTGAACCG
CAGGTTGCCCGTCTGTGCCAGAAAGCGCGTGAAGTTCTGGAAAAAGAAGAAAACGTGCAC
ACCGTTCGTGCGCCAGTTACCGTTTGCGGTGACATCCATGGTCAGTTCCACGATCTGCTG
GAACTCCTGAAAATTGGTGGTCTCCCACCGGACACCAACTATCTCTTCATGGGTGACTAC
GTTGATCGCGGCTACTACTCCGTTGAAACCGTTACGCTCCTCCTGCTGTTCAAAATCCGC
TACCCGGAGCGTGTTCAGATCCTGCGTGGTAATCATGAATCTCGTCAGATCACCCAGGTT
TACGGCTTCTACGACGAATGCGTTCGTAAATACGGTTCTGCGAATGTTTGGAAACTGCTG
ACGGATCTGTTCGACTACCTGCCGCTGGCGGCTGTTGTTGAAAACGAAATCTTCTGCCTG
CACGGTGGCCTGTCTCCGACCCTGGACTCTCTGGCTCACATCCGTTCTCTGGAACGTGTC
CAAGAGGTCCCGCATGAAGGCCCGATGTGCGACCTCCTCTGGTCCGATCCGGAAGACAAA
GACGGTTGGGGTATCTCTCCGCGTGGTGCGGGTTTCACCTTCGGTGCGGACATTACCGAA
CAGTTCTGCCACAACAATGGTCTGAAAACCATCGCGCGTGCTCACCAGCTCGTAGCCGAG
GGTTACTCTTGGGCGCACTCTGATAAACTGGTTACTATCTTCTCTGCGCCGAACTACTGC
TATCGTTGTGGTAACCTGGCGGGTCTGCTCGAACTCGACGAACACATGAACAAATGCTTC
TTTCAGTTCGACCCGGCACCTCGTCGCGGTGAAGCACAAGTCTCCAAGAAGACCCCGGAC
TACTTCCTGTAA
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
SIGMA-ALDRICH RESOURCES
Enzyme Explorer
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer.html
Enzyme Classification Index (EC number)
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=14573088